Phosphoinositide-mediated signaling regulates a broad range of cellular pathways, including sorting of proteins and lipids between organelles, cell proliferation and death (apoptosis), cell motility, and the cytoskeleton. Phosphoinositide 3-kinases (PI3Ks), which phosphorylate the D-3 position of the inositol ring, have been implicated as important regulatory enzymes in each of these pathways. The downstream effects of PI3K signaling are mediated, at least in part, by effector proteins which bind PI3K products directly, including the recently described FYVE sub-family of RING domains. Phosphoinositide (PI) binding by FYVE domains appears to be exquisitely specific; two have been shown to bind phosphatidylinositol 3-phosphate (PtdIns(3)P) nearly exclusively, suggesting that proteins containing FYVE domains are effectors of only a subset of PI3Ks. The long term goal of this research is to understand regulation of protein and membrane trafficking by PI3K in the yeast Saccharomyces cerevisiae. This organism has a single PI3K, encoded by the VPS34 gene, and five proteins containing FYVE domains. The proposed studies focus on four FYVE domain proteins. In Specific Aim 1, biochemical, genetic, and microscopic methods will be used to identify interactions between Vac1p and other components of the Golgi-to-endosome trafficking machinery which are regulated by Vps34 PI3K. In Specific Aim 2, the mechanism by which Vac1p is localized to endosomal organelles will be determined using a combination of cell fractionation and fluorescence microscopy- based methods. There are two currently uncharacterized FYVE domain proteins encoded in the yeast genome, Pib1p and Pib2p. In Specific Aim 3, the intracellular compartments where these proteins are localized will be identified, and trafficking pathways regulated by these proteins will be ientified and characterized. In Specific Aim 4, residues of the human EEA1 FYVE domain required for high affinity PtdIns(3)P binding will be identified. The PI-binding specificities of five other FYVE domain proteins will be determined, and the relative contribution of their FYVE domains to overall PI-binding examined. The proposed studies will contribute to an understanding of how P13Ks regulate vesicle-mediated interorganelle trafficking, and will provide a structure/function framework for understanding the functions of newly discovered FYVE domain proteins.